Gram-negative flagellin is a potent activator of innate immune cells cultured in vitro. Results from our studies as well as those of others indicate that flagellin stimulates innate and adaptive immunity in vivo. We will determine the in vivo mechanisms of flagellin activation of innate and adaptive immunity in the lung. We propose to determine the flagellin-induced events that connect innate and adaptive immune responses to microbial antigens. These studies will establish important principles for use of toll-like receptor agonists as adjuvants to enhance immunity against respiratory pathogens-including those that might be used as agents of bio-terrorism. To that end, we propose the following aims: Specific Aim 1: To determine the mechanisms that regulate the stimulatory effect of flagellin on innate immunity in the mouse lung. We propose to quantify the effect of flagellin on innate immune responses in the mouse lung and delineate the factors that regulate these responses. Specific Aim 2: To define the cellular mechanism(s) for flagellin adjuvant activity in the promotion of a humoral immune response against the bacterial pathogen, Yersinia pestis. We will examine the effect of flagellin on the antibody response in BALB/c mice immunized with a Y. pestis F1N fusion protein. As a test of flagellin adjuvant activity, we will evaluate the ability of flagellin to enhance the ability of F1/V-immunized BALB/c to clear Y. pestis following challenge with bacteria. Specific Aim 3. To define the cellular mechanism(s) for flagellin adjuvant activity in the promotion of a CTL-mediated immune response against respiratory infections with poxviruses. We will determine if flagellin promotes the generation of CTL following immunization with soluble antigen and determine if flagellin promotes protection against viral challenge. In addition to determining the mechanism(s) responsible for the adjuvant activity of flagellin, our studies are dedicated to defining the window of opportunity within which flagellin promotes robust, protective adaptive immunity without triggering an inappropriate level of inflammation-associated tissue damage.